CN110590881A

CN110590881A - Novel oligomeric stilbene compounds in iris lactea kernels and extraction method and application thereof

Info

Publication number: CN110590881A
Application number: CN201910900109.3A
Authority: CN
Inventors: 王洪伦; 铁芳芳
Original assignee: Northwest Institute of Plateau Biology of CAS
Current assignee: Northwest Institute of Plateau Biology of CAS
Priority date: 2019-09-23
Filing date: 2019-09-23
Publication date: 2019-12-20
Anticipated expiration: 2039-09-23
Also published as: CN110590881B

Abstract

The invention discloses a novel oligomeric stilbene compound in Chinese iris seed kernel and an extraction method and application thereof, wherein the alkali extraction acid precipitation of the Chinese iris seed kernel is amplified and prepared, acetonitrile aqueous solution is used as a mobile phase, and the novel oligomeric stilbene compound S11(Vitisin A-13b-o-glucoside) can be obtained by two-time preparation. The invention also provides application of the novel compound S11 in preparation of a medicament for preventing and/or treating lipid metabolism disorder, and activity research on the compound S11 proves that the compound S11 can inhibit cell adipogenic differentiation and lipid generation, so that the compound S11 can be used for preparing the medicament for preventing or treating lipid metabolism disorder diseases such as obesity, hyperlipidemia and the like.

Description

Novel oligomeric stilbene compounds in iris lactea kernels and extraction method and application thereof

Technical Field

The invention relates to an oligomeric stilbene compound in iris lactea seed kernel and the medical application field thereof, in particular to a novel oligomeric stilbene compound in iris lactea seed kernel and an extraction method and application thereof.

Background

Stilbene compounds are the generic name for a class of compounds having a1, 2-stilbene skeleton and polymers composed thereof. Mainly exists in xylem of plants, is originally found in rhubarb of Polygonaceae, and comprises stilbene, diphenyl ethyl, phenanthrene monomer and polymer thereof. The oligomeric stilbene compounds belong to non-flavone polyphenol compounds, and are present in Gentianaceae, Paeoniaceae, Gnetaceae, Rosaceae, Cyperaceae, Leguminosae, Moraceae, Papilionaceae, Iridaceae, Vitaceae, etc. In recent years, researches report that stilbene compounds have various biological activities, such as neuroprotection, antivirus, antibiosis, anti-inflammation, antitumor, antioxidation, anti-AIDS, liver protection and the like.

Iris lactea (Iris lacteal pall. var. chinensis (Fisch.) Koidz.), alias Iris lactea, Indian kalimeris herb, orchid grass and the like are perennial herbaceous perennial plants of Iris (Iris) of Iridaceae of Angiospermae. The Chinese iris is widely distributed in northwest, northeast, east and northeast of China, and simultaneously the Chinese and Western languages, Russia, Mongolia and Korean are also distributed. Chinese iris is a traditional Chinese herbal medicine, according to records in Chinese materia medica, Chinese iris is a whole plant of plant Chinese iris, and is bitter in taste, slightly sweet in nature and slightly cold in nature. It enters kidney, bladder and liver meridians. It has effects of clearing heat and detoxicating, inducing diuresis for treating stranguria, promoting blood circulation and detumescence, and can be used for treating pharyngitis, stranguria with turbid urine, arthralgia, superficial infection and malignant sore, and leaf, flower, root and seed of Iris lactea can also be used as medicine. The Chinese iris flower can be used as a medicine after being dried in the sun, is salty in property, sour, bitter and slightly cool in taste, has the functions of clearing heat and cooling blood, and is used for treating symptoms such as sore throat, hematemesis and the like; the Chinese iris root has mild nature and sweet taste, has the effects of clearing away heat and toxic materials, and can be used for treating toothache, acute infectious hepatitis, pharyngitis, etc.; the seed has effects of clearing heat and detoxicating, promoting diuresis and stopping bleeding, and can be used for treating jaundice, leucorrhea, dysentery, hematemesis, etc. Modern pharmacology indicates that the Chinese iris has various pharmacological effects, such as radiosensitization, biological resistance, immunity enhancement, cancer resistance, glycolipid metabolism improvement and the like.

To date, a variety of chemical components have been isolated from iris lactea including flavonoids, benzoquinones, stilbenes, sterols, volatile oils, and the like.

Stilbenes, are stilbenes or oligomers thereof. However, the stilbene compounds separated from the iris lactea are very little and few in quantity. At present, few reports are reported about the extraction and separation method and the related content measurement research of the stilbene compounds, and the activity research of the related components is also less. If the microorganism is detected, separating r-2-viniferin and trans-epsilon-viniferin from Iris lactea by adopting a 95% ethanol reflux extraction method and a semi-preparative liquid chromatography; luhuanhuan and others adopt high-speed counter-current chromatography to separate and purify the Chinese iris seeds to obtain VitisinA, VitisinB and VitisinC. Therefore, there is a need for further intensive and fine research on other novel chemical components and pharmacological actions of iris lactea and research on the activity mechanism based on the existing research.

Lipid metabolism disorder diseases such as obesity, hypertension, hyperlipidemia, cardiovascular diseases and the like gradually damage the health of people, and long-term taking of traditional treatment medicines can cause side effect influence on the bodies of patients, so that the development of safer and more effective medicines from natural plants is urgently needed.

Disclosure of Invention

The invention provides a novel oligomeric stilbene compound extracted from Chinese iris and application thereof in preparing medicaments for preventing and/or treating lipid metabolism disorder related diseases, wherein the compound has the effects of inhibiting cell adipogenic differentiation and inhibiting cell lipid generation.

The invention provides a novel oligomeric stilbene compound in iris lactea, and the structural formula of the compound is shown as a formula S11:

the existing extraction and separation method for the active substances in the iris lactea mostly needs a large amount of organic solvents such as methanol, ethanol, acetone, ethyl acetate and the like, but the organic solvents are high in price, volatile and poor in production safety. The stilbene compounds structurally have phenolic hydroxyl groups, have certain weak acidity, can be combined with alkali to improve the water solubility, but are difficult to dissolve in an acidic solution. The present invention extracts and separates Chinese iris through alkali extraction and acid precipitation, and through one-dimensional and two-dimensional nuclear magnetic identification, one new stilbene compound S11 named Vitisin A-13b-O-glucoside is obtained.

The invention also provides a preparation method of the compound S11, which comprises the following steps:

(1) preparing semen Iridis seed extract by preparative chromatography to obtain component S1;

wherein the preparative chromatographic conditions comprise:

a chromatographic column: a C18 chromatography column; preferably the specification is 20mm x 250mm, 10 μm;

mobile phase: 5-30% acetonitrile water solution; gradient elution was performed using the following procedure: 0min, 5-10% acetonitrile water solution; 35-45 min, 20-30% acetonitrile water solution; further, gradient elution was performed using the following procedure: 0min, 5% acetonitrile water solution; 40min, 30% acetonitrile in water;

taking a characteristic peak with the highest peak height in the chromatogram as a reference peak (40.593 min in figure 1), wherein relative retention time of the component S1 is 0.71-0.77, preferably 0.74-0.75;

the relative retention time of S1 is: taking the component S4 as a reference component, taking the retention time of the component S4 as a denominator, taking the retention time of the component S1 as a numerator, and taking the ratio of the retention time of the component S4 as the relative retention time of the component S1, namely: the relative retention time of S1 ═ retention time of S1/retention time of S4.

In one embodiment of the invention, the component S1 is a component with a retention time of 29-31 min, and more specifically, the component S1 is a component with a retention time of 30.333 min.

(2) Semi-preparative separation of the component S1 to give compound S11;

wherein the conditions for the semi-preparative separation comprise:

mobile phase: 20-30% acetonitrile water solution; isocratic elution.

S11 is the most intense peak in the preparative chromatogram.

In a particular embodiment of the invention, the preparative chromatography or semi-preparative separation further comprises at least one of the following conditions:

detection wavelength: 210 nm;

column temperature: 25-35 ℃;

flow rate: 40-50 mL/min.

In a specific embodiment of the invention, the Chinese iris seed kernel extract is a Chinese iris seed kernel alkali acid-extracted precipitate.

Further, the preparation method of the irisquinone seed kernel alkali acid-extraction precipitate comprises the following steps:

firstly, extracting the Chinese iris seed kernels with an alkali solution to obtain an alkali extracting solution;

adding acid into the alkali extracting solution until the precipitate is completely generated, and collecting the precipitate;

dissolving the precipitate in alcohol, and removing the solvent to obtain alkali-extracted acid precipitate of Chinese iris seed; the alcohol is selected from methanol and/or ethanol.

Further, the alkali solution is a NaOH solution with the mass fraction of 1-10%, and preferably a NaOH solution with the mass fraction of 5%.

Further, the addition of acid to precipitate completely adjusts the pH of the solution to not more than 3 by the addition of acid.

Further, the acid is selected from dilute sulfuric acid and/or hydrochloric acid.

The dilute sulfuric acid is an aqueous solution of sulfuric acid with a solute mass fraction of less than or equal to 70%, and the concentration of the dilute sulfuric acid is not further limited in the invention as long as experimental conditions can be met, and the hydrochloric acid is the same.

Further, the ethanol is an ethanol water solution with the volume concentration of 95-100%.

The ethanol is an ethanol aqueous solution with the volume concentration of 95-100%, and the ethanol can be an ethanol aqueous solution with the volume concentration of more than or equal to 95% or can be pure ethanol. In practice, 95% ethanol, i.e. 95% strength by volume ethanol in water, is often used.

Further, in the step (1), the irisquinone seed kernel alkali extraction acid precipitate is dissolved in methanol, and the obtained methanol extraction liquid is sampled.

The methanol extract of the alkali extraction acid precipitate of the iris lactea seed can be obtained by referring to various solvent dissolving and extracting modes in the prior art. In the specific embodiment of the invention, the irisquinone seed kernel alkali extraction acid precipitate is dissolved in methanol, and then filtered supernatant obtained by filtering through an organic membrane is the methanol extracting solution of the irisquinone seed kernel alkali extraction acid precipitate.

Further, the feed-liquid ratio of the irisquinone seed alkali-acid-extraction precipitate to the methanol is 0.2-1 g/ml; furthermore, the feed-liquid ratio of the irisquinone seed kernel alkali acid-extraction sediment to the methanol is 0.4 g/ml.

The invention provides application of the oligomeric stilbene compounds in preparing products for preventing and/or treating lipid metabolism disorder.

The C2C12 cell is a murine muscle precursor cell line, and is an ideal cell model for studying muscle growth and development, muscle cell differentiation and lipid metabolism. Since both adipocytes and muscle cells originate from embryonic stem cells, muscle cells can be transdifferentiated into adipocytes or adipocyte-like cells (adipocyte-like cells) under specific conditions. For example, under the condition of drug stimulation or environmental change, the C2C12 cell loses the myogenic property of muscle cells and is transformed into fat cells, a large number of fat droplets are generated in the cells, and fat cell specific genes are expressed. The invention researches the influence of Vitisin A-13b-o-glucoside on the lipogenic transdifferentiation of C2C12 and provides a certain experimental basis for improving the lipometabolism.

Products described in the present invention include, but are not limited to, pharmaceuticals, nutraceuticals, foods, and the like.

Further, the product is a product with lipid-lowering effect, such as lipid-lowering drugs and the like.

The product or the lipid-lowering medicine with the lipid-lowering effect is a product or a medicine for inhibiting the generation of lipid drops in fat cell cytoplasm and reducing the content of triglyceride.

The products can be divided into two categories, namely inhibition of cell lipogenic transdifferentiation and inhibition of mature adipocyte lipogenesis according to the cell differentiation and maturation.

Further, the product for preventing and/or treating lipid metabolism disorder includes a product for inhibiting cell adipogenic differentiation and/or a product for inhibiting cell lipogenesis; the product can be medicine, health product or other product with cell lipogenesis transdifferentiation inhibiting effect and/or cell lipogenesis inhibiting effect.

When the product is a medicament, the medicament comprises at least one of prevention and/or treatment of obesity, hypertension, hyperlipidemia, cardiovascular diseases and metabolic syndrome related diseases.

Obesity, hypertension, hyperlipidemia, cardiovascular diseases, metabolic syndrome, etc. are closely related to the content of plasma lipids such as Triglyceride (TG), Free Cholesterol (FC), Cholesterol Ester (CE) and phospholipids, and when plasma lipids are reduced to a certain concentration range in vivo, these diseases can be effectively controlled or treated. Experiments prove that the compound S11 can effectively inhibit lipid drop accumulation, reduce TG content, and regulate gene expression level and related protein expression level of adipocyte transcription factor through related signal pathways, so as to inhibit adipogenic differentiation and lipid generation of cells, and can be used for preparing products for preventing and/or treating obesity, hypertension, hyperlipidemia, cardiovascular diseases and metabolic syndrome related diseases.

The invention also provides application of the oligomeric stilbene compounds in preparing at least one of PPAR gamma antagonists, C/EBP alpha antagonists, FAS inhibitors and ACC inhibitors.

The PPAR gamma antagonist, the C/EBP alpha antagonist, the FAS inhibitor and the ACC inhibitor are drugs for reducing the gene expression of adipocyte transcription factors PPAR gamma and C/EBP alpha and the protein expression level of FAS and ACC.

Differentiation of undifferentiated cells into mature adipocytes requires a series of sophisticated transcription factor regulation, PPAR γ is an essential regulatory factor for adipogenesis, and is highly expressed at the early stage of adipogenesis; C/EBP α, the so-called CCAAT/enhancer binding protein α, is abundantly expressed during the metaphase stage of adipocyte differentiation.

FAS is a fatty acid synthase that plays an important role in lipid production; ACC is acetyl-coa carboxylase, the rate-limiting enzyme in the synthesis of fatty acids.

The compound S11 can reduce the gene expression of cell transcription factors PPAR gamma and C/EBP alpha and the protein expression level of FAS and ACC, thereby effectively inhibiting the products of cell adipogenic differentiation and inhibiting the generation of cell lipid, and further being used for preparing medicines for preventing and/or treating diseases related to lipid metabolism disorder, such as obesity, hypertension, hyperlipidemia, cardiovascular diseases and the like.

The invention also provides a lipid-lowering product, which comprises the compound S11.

The invention has the beneficial effects that:

(1) the invention adopts alkali extraction and acid precipitation combined with semi-preparative liquid chromatograph to rapidly and efficiently separate and prepare a brand new oligomeric stilbene compound S11, and more comprehensively develops the active substance of the Chinese iris.

(2) The invention extracts a novel oligomeric stilbene compound from iris lactea seeds and performs activity research on the oligomeric stilbene compound, and the result shows that the compound S11 can inhibit lipid droplet accumulation, reduce TG content, and regulate the gene expression level and the related protein expression level of adipocyte transcription factors through related signal pathways, so that adipogenic differentiation and lipid generation of cells are inhibited, and a powerful direction is provided for researching and applying to lipid metabolism disorder diseases such as obesity, cardiovascular diseases and the like.

(3) The method provided by the invention more comprehensively excavates the medicinal value of the iris lactea, expands the clinical application of the iris lactea, and provides more reference bases for developing potential plant-derived medicines for treating diseases related to lipid metabolism disorder.

Drawings

FIG. 1 is a separation chromatogram of component S1 on a preparative chromatographic column;

FIG. 2 is a preparation diagram of component S11 on a preparative chromatographic column;

FIG. 3 is a purity analysis chart of component S11;

FIG. 4 is a nuclear magnetic H spectrum of novel compound S11;

FIG. 5 is a nuclear magnetic C spectrum of novel compound S11;

FIG. 6 is a HSQC plot of novel compound S11;

FIG. 7 is a HMBC diagram of novel compound S11;

FIG. 8 is a HHCOSY plot of novel compound S11;

FIG. 9 is a NOESY diagram of novel compound S11;

FIG. 10 is a graph of the effect of different concentrations of ViisinA-13 b-o-glucoside on cell viability;

FIG. 11 shows the accumulation of intracellular lipid droplets from C2C12 cells during the induction of adipogenesis at different times (100X);

FIG. 12 is the intracellular triglyceride content of C2C12 cells during different times of induced adipogenesis;

FIG. 13 is a graph of the effect of ViisinA-13 b-o-glucoside on lipid droplet production by C2C12 cells;

FIG. 14 is a graph of the effect of ViisinA-13 b-o-glucoside on triglyceride content of C2C12 cells;

FIG. 15 is the expression levels of the C2C12 cell lipogenesis associated proteins during induction of adipogenesis at different times;

FIG. 16 is the expression level of proteins involved in lipid degradation of C2C12 cells during induction of adipogenesis at different times;

FIG. 17 is a graph of the effect of ViisinA-13 b-o-glucoside on the expression of C2C12 cell transcription factor;

FIG. 18 is a graph of the effect of ViisinA-13 b-o-glucoside on the expression of C2C12 cell lipogenesis associated proteins.

Detailed Description

The preparation and application of the novel oligomeric stilbene compounds of the invention are further illustrated by the following specific examples and specific experiments, and the effects of the novel oligomeric stilbene compounds in inhibiting cell lipid differentiation and lipid generation are verified and illustrated.

The instrument comprises the following steps: agilent 1260 series high performance liquid chromatograph equipped with G1311C quaternary gradient pump, G1329B autosampler, G1316A column oven, G1315D detector; hanbang NP7000C high performance liquid chromatography, RE52-98 rotary evaporator (Shanghai Yangrong Biochemical apparatus factory).

Reagent: the analytical and preparative acetonitrile is from new blue view chemical industry of Yunnan, chemical industry, etc., the water for chromatography is Wahaha purified water, and the organic solvent applied to HPLC is pure chromatography.

In the embodiment of the invention, the method for detecting the purity of the compound is obtained by using HPLC through an area normalization method, and the calculation mode is as follows: target compound purity% (% target compound peak area/total peak area)% 100%

EXAMPLE 1 preparation of novel Oligostilbene Compounds

(1) Washing the selected Chinese iris seed after removing impurities with pure water, drying in the shade, peeling, and crushing to 30 meshes to obtain a Chinese iris seed kernel raw material;

(2) soaking 22kg of semen Iridis seed kernel in 5% NaOH solution at normal temperature for 12 hr, wherein the 5% NaOH solution is 3kg NaOH solution dissolved in 60L ultrapure water, filtering the extractive solution, and collecting;

(3) adding dilute H dropwise into the extracting solution obtained in the step (2)₂SO₄Adjusting the pH value of the solution to 3, and standing the solution at room temperature for 1h to precipitate;

(4) collecting the precipitate generated in the step (3) and centrifuging at room temperature, wherein the centrifugation conditions are as follows: 4500rpm for 10min, and collecting the obtained precipitate;

(5) dissolving the acid precipitate collected in the step (4) with 12L of 95% ethanol to fully dissolve the acid precipitate;

(6) concentrating and evaporating the ethanol solution obtained in the step (5) under reduced pressure, wherein the separation pressure is 1.0 MPa; the separation temperature is 25-30 ℃; drying at 65 deg.C to obtain alkali-extracted acid precipitate of semen Iridis;

(7) dissolving 20g of the irisquinone seed kernel alkali extraction acid precipitate obtained in the step (6) in 50mL of methanol solution, and filtering by adopting an organic membrane to obtain clear filtered sample liquid;

(8) amplifying the clarified filtered sample liquid obtained in the step (7) on a DAC50 preparation column, wherein the mobile phase is as follows: 5-30% acetonitrile water solution; gradient elution was performed using the following procedure: 0min, 5% acetonitrile water solution; 40min, 30% acetonitrile in water; loading 1mL, column: hedera C18, 20 x 250mm, 10 μm packing, flow rate: 40 mL/min; column temperature: 35 ℃; detection wavelength: 210 nm. Four components S1, S2, S3, S4 were obtained as shown in FIG. 1.

(9) Dissolving the S1 component (the component corresponding to the retention time of 29-31 min; more specifically, the component corresponding to the peak value of 30.333 min) obtained in the step (8) with methanol, and finally selecting 30% of acetonitrile for isocratic elution through optimization of preparation conditions, wherein the chromatographic column is a C18 semi-preparative chromatographic column with 20 x 250mm, the packing with 10 mu m, and the flow rate is as follows: 40 mL/min; column temperature: 35 ℃; detection wavelength: 210nm, and thus gave sample S11 (retention time 26.609min), as shown in FIG. 2.

(10) And (3) carrying out high performance liquid chromatography detection on the sample S11 obtained in the step (9) to determine the purity, wherein the detection conditions are as follows: fluidity: 10-50% acetonitrile water, detection time: 0-40 min, Kromasil C18, 5 μm, 4.6 × 250mm, detection wavelength 210 nm.

The purity of the compound S11(Vitisin A-13b-O-glucoside) prepared by the method reaches 91.11% by HPLC detection, as shown in FIG. 3. The structure of S11 is as follows:

the results of structural (nuclear magnetic data) confirmation of compound S11 are shown in table 1 and fig. 4 to 9:

table 1.¹H and ¹³C NMR spectroscopic data for S11(CD₃OD,δ_H:3.30ppm,δ_C:49.0ppm)

EXAMPLE 2 preparation of New Oligostilbene Compounds

(2) soaking 22kg of Chinese iris seed kernels with 10% NaOH solution at normal temperature for 8h, wherein the 10% NaOH solution is 6kg of NaOH solution dissolved in 60L of ultrapure water, and filtering and collecting the extract;

(3) dropwise adding hydrochloric acid into the extracting solution obtained in the step (2), adjusting the pH value to be 3, and standing at room temperature for 1h to generate precipitate;

(5) dissolving the acid precipitate collected in the step (4) with 12L of methanol to fully dissolve the acid precipitate;

(8) amplifying the clarified filtered sample liquid obtained in the step (7) on a DAC50 preparation column, wherein the mobile phase is as follows: 10-30% acetonitrile water solution; gradient elution was performed using the following procedure: 0min, 10% acetonitrile water solution; 35min, 30% acetonitrile water solution; loading 1mL, column: hedera C18, 20 x 250mm, 10 μm packing, flow rate: 50 mL/min; column temperature: 25 ℃; detection wavelength: 210 nm. Four components S1, S2, S3, S4 were obtained.

(9) Dissolving the S1 fraction obtained in the step (8) with methanol, and finally selecting 30% acetonitrile for isocratic elution through optimizing preparation conditions, wherein the chromatographic column is a 20 x 250mm C18 semi-preparative chromatographic column, a 10-micron filler, and the flow rate is as follows: 50 mL/min; column temperature: 25 ℃; detection wavelength: 210nm, and thereby yielded sample S11.

The purity of the compound S11(Vitisin A-13b-O-glucoside) prepared by the method reaches 92.05% through HPLC detection.

EXAMPLE 3 preparation of novel Oligostilbene Compounds

(2) soaking 22kg of semen Iridis seed kernel in 1% NaOH solution at normal temperature for 18h, wherein the 1% NaOH solution is 0.6kg NaOH dissolved in 60L ultrapure water, filtering the extractive solution, and collecting;

(3) to the product obtained in step (2)Adding diluted H dropwise into the extractive solution₂SO₄Adjusting the pH value of the solution to 3, and standing the solution at room temperature for 1h to precipitate;

(8) amplifying the clarified filtered sample liquid obtained in the step (7) on a DAC50 preparation column, wherein the mobile phase is as follows: 5-20% acetonitrile water solution; gradient elution was performed using the following procedure: 0min, 5% acetonitrile water solution; 45min, 20% acetonitrile water solution; loading 1mL, column: hedera C18, 20 x 250mm, 10 μm packing, flow rate: 45 mL/min; column temperature: 30 ℃; detection wavelength: 210 nm. Four components S1, S2, S3, S4 were obtained.

(9) Dissolving the S1 fraction obtained in the step (8) with methanol, and finally selecting 30% acetonitrile for isocratic elution through optimizing preparation conditions, wherein the chromatographic column is a 20 x 250mm C18 semi-preparative chromatographic column, a 10-micron filler, and the flow rate is as follows: 45 mL/min; column temperature: 30 ℃; detection wavelength: 210nm, and thereby yielded sample S11.

The purity of the compound S11(Vitisin A-13b-O-glucoside) prepared by the method reaches 91.08 percent through HPLC detection.

The advantageous effect of the compound S11 according to the invention is demonstrated by the following test examples:

test example 1

Experimental materials and Experimental preparations

Myoblasts C2C12 were purchased from shanghai life science research institute of chinese academy of sciences. The compound VitisinA-13b-o-glucoside was isolated and purified by the method of example 1.

Peptide bovine serum was purchased from Gibco, usa; DMEM high-glucose medium, trypsin and PBS were purchased from Hyclone, usa; insulin, dexamethasone, rosiglitazone, methylisobutylxanthine and oil red O powders were all purchased from Sigma, usa. Both primary and secondary antibodies used for western blot analysis were purchased from Cell Signaling Technology (CST) corporation.

Statistical analysis: the test data is represented by 'mean value plus or minus standard deviation', the experimental result is subjected to one-factor variance analysis by Graphid7.0 analysis software, P <0.05 is taken as a significant difference, and P <0.01 is taken as a very significant difference.

First, C2C12 cell induces lipotropy and transdifferentiation

C2C12 myoblasts grow to confluence and differentiate automatically into myocytes. To mimic the process of in vivo intraadipose fat deposition, this assay was treated with fused C2C12 cells induced by a classical tripartite inducer consisting of insulin, dexamethasone, and methylisobutylpurin.

Inducing and differentiating the C2C12 cells by adopting a cocktail method according to the following steps: the C2C12 cells in good cell state were seeded on a culture plate at a plating density of 5X 10⁴Culturing with high-glucose DMEM culture solution containing 10% FBS until the cell density reaches about 80%, discarding the complete culture solution, adding culture solution containing 10 μ g/mL of insulin, 0.5mM of 3-isobutyl-1-methylxanthine, 1 μ M of dexamethasone and 10 μ M of rosiglitazone as adipogenic differentiation inducer I, culturing for two days, then changing to culture solution containing 10 μ g/mL of insulin and 10 μ M of rosiglitazone as inducer II, culturing for two days, then changing to normal culture, and changing the culture solution every other day. At day 8 of induction, more than 85% of the cells in the cells were observed to assume the form of mature adipocytes, i.e., they were microscopically found to have lipid droplets of varying sizes. Cells were harvested at 0, 2, 6 and 8 days of induction, respectively.

Second, cell viability assay

The cell viability is measured by an SRB method, and the specific steps are as follows: (1) the cells were cultured at 5X 10⁴Is inoculated into a 96-well plate with a medium containing 1Culturing in 0% FBS high-glucose DMEM culture solution until the cell growth reaches about 50-60%, adding the monomer compound to be tested, and incubating for 48h when the cell density is 50-60% and the compound (each compound has 5 concentration gradients); (2) after the drug treatment, the stock solution is discarded, 100 μ L of fresh DMEM is added into each well, 25 μ L of 50% TCA (in dark place, can be added by a row gun) is added, the mixture is placed for 5min at room temperature, and the mixture is placed for 1h at 4 ℃; (3) discarding stock solution, using flowing ddH₂And washing each hole for 5 times, and forcibly throwing out after washing each time. Drying in a superclean bench for 15min (high wind); (4) add 70. mu.L of 0.4% SRB to each well and shake for 30min (protected from light). (5) The SRB dye solution is discarded, 1% glacial acetic acid is added into each hole for washing for 4 times, and the SRB dye solution is forcibly thrown out after washing for each time. Drying in a superclean bench for 15min (high wind); (6) mu.L of 100mM Tribase was added to each well for solubilization and incubated at 37 ℃ for 20min with shaking. (7) OD was measured with a microplate reader at a wavelength of 540 nm. Cell viability was calculated according to the formula:

cell viability (%). ratio (drug OD/blank OD). times.100%

The results are shown in FIG. 10. When the concentration of ViisinA-13 b-o-glucoside is 1. mu.M, the activity of the cells is not affected at all, and the activity of the cells is close to 100 percent. When the concentration is 10. mu.M or more than 10. mu.M, the viability of the cells gradually decreases with increasing concentration of the compound. Therefore, in the subsequent experiments, we chose 5 μ M as the optimal concentration of the compound.

Thirdly, the accumulation condition of intracellular lipid drops of C2C12 cells and the content of triglyceride in the cells at different induction times

The sterile cover slips were placed in a 24-well plate, 1mL of DMEM was added, and the plate was incubated in an incubator for 30 min. The medium was aspirated and the cells were plated at 5X 10⁴Inoculating each/mL of the cells on a slide, adding 1mL of cell suspension into each hole, pressing the slide by using a needle, removing air bubbles below the slide, placing the slide in an incubator for culture, performing induced differentiation when the cell density reaches about 80%, performing the induced differentiation process as described in 1.3, fixing after the induction is finished, adding a pre-prepared oil red O working solution to dye the cell surface, and standing for 60min in a dark place. The cells were washed with 70% ethanol, excess dye was removed, washed 3-4 times with double distilled water, observed under a microscope and photographed.

C2C12 cells were cultured at 5X 10⁴Inoculating the cells/well into a 24-well plate, inducing and differentiating when the cell density reaches about 80%, wherein the inducing process is as described in 1.3, and measuring the TG content on the 8 th day of induction by the specific method as follows: (1) cell treatment: on day 8 of induction, cell culture fluid was aspirated, washed twice with cold PBS, and cell lysate PIPA was added for 5min lysis (this operation was performed on ice) (2) cell collection: after cell lysis, cells were transferred from the bottom of the well plate to a 1.5mL centrifuge tube using a cell scraper to prepare a homogenate for direct assay without centrifugation. (3) Adding 2 mu L of sample to be detected into each hole of a 96-hole plate, adding 2 mu L of distilled water into a blank hole, adding 2 mu L of standard substance into a standard hole, then adding 160 mu L of reagent R1 into each hole, uniformly mixing, incubating for 5min at 37 ℃, and measuring the A1 value by using an enzyme-labeling instrument under the condition of 546nm wavelength; immediately thereafter, 40. mu.L of reagent R2 was added to each sample, mixed well, incubated at 37 ℃ for 5min and the second absorbance A2 was read. Change in absorbance of each tube_△A is A2-A1. And finally, determining the protein concentration in the sample to be detected by using a BCA method, and correcting.

TG content ═ OD_{Sample (I)}-OD_{Blank space})/(OD_Proof-OD_{Blank space}) X calibrator concentration (mM)/protein concentration of sample to be tested (gprot/L)

C2C12 myoblasts grow to confluence and differentiate automatically into myocytes. To simulate the process of in vivo fat deposition, the assay induced fused C2C12 cells with a classical tripartite inducer consisting of insulin, dexamethasone, and methylisobutylpurin, and the cells were collected on days 0, 2, 4, and 8 of treatment for oil red O staining and determination of triglyceride content, respectively. The results showed that myotubes appeared from day 4, and some of the myotubes contained lipid droplets (fig. 11); as the induction time increased, intracellular lipid droplets gradually increased until day 8, where they accumulated most and were abundant (fig. 11). The results of measurement of triglyceride levels are shown in FIG. 12, in which the intracellular triglyceride levels were different depending on the induction time, and were low at day 0, but increased gradually with the increase in induction time from the second day of induction until the highest intracellular triglyceride level was reached at day 8.

1. Effect of VitisinA-13b-o-glucoside on lipid droplet production by C2C12 cells

C2C12 cells were induced to undergo adipogenic transformation according to the method of "one, C2C12 cells induced to undergo lipogenic transdifferentiation" in this test example, and 5mM Vitisin-13b-o-glucoside was added simultaneously with the addition of the inducing solution. By day 8 of induced differentiation, oil red O staining revealed that no lipid droplets were formed in the undifferentiated cells and that a large number of lipid droplets were present in the differentiated cells. After the differentiation group was treated with vitasin-13 b-o-glucoside, the vitasin-13 b-o-glucoside group inhibited the formation of lipid droplets, as shown in fig. 13.

2. Effect of VitisinA-13b-o-glucoside on triglyceride content of C2C12 cells

C2C12 cells were induced to adipogenic according to the method of 1.3, and 5mM Vitisin-13b-o-glucoside was added simultaneously with the addition of the inducing solution. Cells were collected by day 8 of induced differentiation and triglyceride content was determined according to the instructions of the kit. As a result, it was found that the content of triglyceride in the undifferentiated group was extremely low and the content of triglyceride in the differentiated group was high. After the treatment of the differentiation group with vitasin-13 b-o-glucoside, the content of triglyceride in the vitasin-13 b-o-glucoside group was decreased compared to the differentiation group, as shown in FIG. 14.

Effects of C2C12 cell lipid metabolism-related protein expression

C2C12 cells were cultured at 5X 10⁴Inoculating each cell/well in a 6-well plate, inducing and differentiating when the cells completely grow to about 80%, wherein the inducing process is as described in 1.3, collecting the cells on the 8 th day of induction, extracting cytoplasmic protein, and performing protein electrophoresis, and the specific method comprises the following steps:

(1) extraction of Total cellular proteins

After the cell is treated by the medicine, the cell is collected and total protein is extracted, the experimental operation is a specific method reference, and finally the extracted total protein is stored at minus 80 ℃.

(2) BCA method for determining protein content

Taking out the frozen protein sample, diluting the protein standard substance to 0.5mg/mL by using PBS (phosphate buffer solution) according to the requirements of the BCA protein quantification kit specification, and mixing the components in the reagent A: and uniformly mixing the reagent B to prepare working solution. Standards and PBS were added to 96-well plates at 20 μ L per well, typically 3 duplicate wells per concentration, as shown in table 2, and used to generate a standard curve. Protein samples were added to a 96-well plate at 1. mu.L, and PBS was added to make up to 20. mu.L. Adding 200 mu LBCA working solution into each hole, fully and uniformly mixing, carrying out oscillation incubation on the sample for 30min at 37 ℃ in a microplate incubation oscillator, measuring a photometric value at 562nm of a microplate reader, and calculating the protein concentration of the sample according to a standard curve. After the protein concentration is determined, adding SDS-PAGE sample buffer into 30 mu g of protein sample, fully mixing, boiling in boiling water for 10min to completely denature the protein. Before loading the protein, the sample is used after being centrifuged and mixed evenly.

TABLE 2BCA method protein quantitation Table

(ii) SDS-PAGE electrophoresis

The specific operation method of protein electrophoresis is the same as the conventional method in the prior art, the proper separation gel concentration is selected according to the molecular weight of the target protein and the table 4, the proper solution system is prepared according to the gel preparation description table 3, the upper layer 5% of concentrated gel is prepared according to the table 5, and electrophoresis is carried out as soon as possible after the gel preparation is finished, wherein the electrophoresis conditions are as follows: stage S1, 80V, 30 min; stage S2, 120V, about 2h, until bromophenol blue in buffer runs to the lowest end of the separation gel, ending electrophoresis.

TABLE 3 optimal separation Range of SDS-PAGE gels at different concentrations

Table 1.2The separation ranges of SDS-PAGE gel with different concentration

TABLE 4 volume of each component required to formulate different volume 10% SDS-PAGE gels

TABLE 5 formulation of the various components required for different volumes of 5% SDS-PAGE gel concentrates

② rotating the membrane

The specific experimental operation method is the same as the conventional method in the prior art when the membrane is switched immediately after the protein electrophoresis is finished.

③ seal

After the transfer, the PVDF membrane was quickly removed from the transfer plate gently with tweezers, rinsed in TBST buffer for 5min, and immediately blocked with 5% skimmed milk powder in TBST for 1h on a shaker at room temperature.

Tetra anti-incubation

After blocking, the PVDF membrane was placed in the diluted primary antibody solution and incubated overnight at 4 ℃ on a horizontal shaker with slow shaking.

Fifthly, incubation with secondary antibody

After the primary antibody incubation is finished and the membrane washing is finished, the PVDF membrane is placed in a solution containing a secondary mouse antibody or a secondary rabbit antibody marked by HRP and incubated for 1h in a shaking table at room temperature.

Sixthly, developing

After all the antibodies are incubated, an ECL method is adopted for detection, a developing solution is prepared according to the proportion of 1:1, mixed ECL is added on a PVDF membrane for luminescence, and a gel imaging system is used for photographing.

1. Expression level of lipogenesis-related protein of C2C12 cells at different induction times

The expression levels of the C2C12 cell lipogenesis-related protein at days 0, 2, 4 and 8 of the different induction times are shown in fig. 15. The levels of ACC, FAS, PPAR γ and C/EBP α protein expression were low in the non-induced C2C12 cells, with PPAR γ being hardly expressed. On days 2, 4 and 8 of induction, the expression levels of ACC, FAS, PPAR γ and C/EBP α proteins in C2C12 cells increased gradually and reached the highest expression values on day 8.

2. Expression level of protein related to lipid degradation of C2C12 cells at different induction times

The expression levels of the proteins involved in lipid degradation of C2C12 cells at days 0, 2, 4 and 8 of different induction times are shown in FIG. 16. The highest level of ACOX was observed in non-induced C2C12 cells, whereas CPT1A, HSL and ATGL proteins were expressed at lower levels, with HSL and ATGL being hardly expressed. On days 2, 4 and 8 of induction, CPT1A, HSL and ATGLA protein expression levels gradually increased in C2C12 cells and reached the highest expression level on day 8, accelerating the rate of triglyceride hydrolysis and fatty acid oxidation.

3. Effect of VitisinA-13b-o-glucoside on transcription factor expression during C2C12 cell lipid differentiation

As shown in fig. 17, the transcription factor PPAR γ associated with adipogenic differentiation in undifferentiated cells was hardly expressed and the expression level was extremely low; PPAR gamma protein is highly expressed in cells of a differentiated group, and the expression content of a transcription factor PPAR gamma in the cells is reduced after 8 days of treatment by simultaneously administering the compound vitasin A-13b-o-glucoside in differentiated C2C12 cells. The transcription factor C/EBP alpha is also expressed in undifferentiated cells, the expression content in differentiated cells reaches the highest, and the expression content of the transcription factor C/EBP alpha in cells is reduced after the compound vitasinA-13 b-o-glucoside is treated. The result shows that the compound vitasin A-13b-o-glucoside has the capability of inhibiting lipogenic transdifferentiation of C2C12 cells.

4. Effect of VitisinA-13b-o-glucoside on expression of C2C12 cell lipogenesis associated protein

FAS is a fatty acid synthase that plays an important role in lipid production; ACC is acetyl-coa carboxylase, the rate-limiting enzyme in the synthesis of fatty acids. As shown in fig. 18, FAS and ACC were hardly expressed in undifferentiated cells, and the expression content was low; a large number of lipid droplets appear in the differentiation group, lipid synthesis is accelerated, and therefore FAS and ACC proteins are highly expressed, and the expression amount reaches the highest value. And after 8 days of treatment with compound vitasin A-13b-o-glucoside administered simultaneously in differentiated C2C12 cells, the intracellular expression levels of FAS and ACC proteins decreased. The result shows that the compound vitasin A-13b-o-glucoside has the capability of inhibiting the generation of C2C12 cell lipid.

In conclusion, the novel oligomeric stilbene compounds extracted by the invention can effectively inhibit cell adipogenic differentiation and lipid generation, and provide more reference basis for developing potential plant-derived medicaments for treating diseases related to lipid metabolism disorder.

The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications of equivalent structures and equivalent processes performed by the present specification and drawings, or directly or indirectly applied to other related technical fields, are included in the scope of the present invention.

Claims

1. An oligomeric stilbene compound, which is characterized by having a structure shown as a formula S11:

2. a process for the preparation of a compound according to claim 1, comprising:

wherein, the conditions of the preparative chromatography comprise:

taking a characteristic peak with the highest peak height in the chromatogram as a reference peak, and relative to the reference peak, the relative retention time of the component S1 is 0.71-0.77, preferably 0.74-0.75;

(2) semi-preparative separation of the component S1 to give compound S11;

wherein the conditions for the semi-preparative separation comprise:

mobile phase: 20-30% acetonitrile water solution; isocratic elution.

Further, the preparative chromatography or semi-preparative separation further comprises at least one of the following conditions:

detection wavelength: 210 nm;

column temperature: 25-35 ℃;

flow rate: 40-50 mL/min.

3. The method according to claim 2, wherein the Iris lactea seed extract is an Iris lactea seed alkali acid-extraction precipitate; further, the preparation method of the irisquinone seed kernel alkali acid-extraction precipitate comprises the following steps:

Further, the alkali solution is a NaOH solution with the mass fraction of 1-10%, preferably a NaOH solution with the mass fraction of 5%;

further, the adding of acid until precipitation is completed is the adding of acid to adjust the pH value of the solution to be not more than 3; further, the acid is selected from dilute sulfuric acid and/or hydrochloric acid;

4. The preparation method according to claim 3, wherein in step (1), the irisquinone seed kernel alkali-extraction acid precipitate is dissolved in methanol, and the obtained methanol extract is loaded;

5. Use of the compound of claim 1 for the preparation of a product for the prevention and/or treatment of a disease associated with a disorder of lipid metabolism.

6. The use according to claim 5, wherein the product is a product with lipid lowering effect.

7. The use according to claim 5, wherein the product is a product inhibiting adipogenic differentiation and/or inhibiting lipogenesis in cells.

8. The use according to claim 5, wherein the product is at least one of a product for preventing and/or treating obesity, hypertension, hyperlipidemia, cardiovascular disease, metabolic syndrome-related disease.

9. Use of a compound according to claim 1 for the preparation of at least one of a PPAR γ antagonist, a C/ebpa antagonist, a FAS inhibitor, and an ACC inhibitor.

10. A lipid lowering product comprising compound S11 of claim 1.